Optogenetics and chemo-optogenetics are powerful tools for modulating cell activities with light. These tools accelerate neuroscience research by providing the necessary means for interrogating neural circuit function. Clinical trials of optogenetic therapy for retinal diseases are already underway. Some of the limitations of these current tools include a generally small light-induced current, their limited ability to manipulate specific cell activity in deep tissue, the need for robust transgene expression to illicit physiological effects, and safety concerns over long-term exogenous transgene expression. The novel chemo-optogenetic tools I develop will address many of these issues. I previously developed a novel chemo-optogenetic tool based on the high conductance TRPA1 channel which is suitable for modulating both neuronal and non-neuronal cell activity in vivo. I have also developed and performed a small molecule screen based on the zebrafish light-induced motion response and discovered molecular photoswitches that target endogenous vertebrate proteins. I am characterizing two hits identified from this screen (Aim 1, K99 phase). One is a step-function chemo- optogenetic system based on the TRPA1 channel. This new system will allow for light-controlled channel ON/OFF, further enhancing TRPA1 utility. The second is a chemo-optogenetic system based on the TRPV1 channel. The next phase of my chemo-optogenetic tool-development program is to enhance TRPA1 channel selectivity for sodium while preserving its high channel conductance (Aim 2, K99/R00 phase). This will provide a more physiologically relevant light-induced generation of action potentials. I will also extend the zebrafish light-induced motion response screening assay to specifically identify endogenous protein-targeting molecular photoswitches with spectra in the near infrared range (Aim 3, R00 phase). The use of near infrared light allows for deeper penetration into tissues and for compatibility with existing optogenetic tools and biosensor imaging. Overall, my proposed research will generate novel chemo-optogenetic tools with improvements to unitary channel conductance, light-controlled ON/OFF activity in deeper tissue, and require no or low levels of exogenous gene expression. My research will also create a platform for the discovery of novel chemo- optogenetic actuators that mimic natural cell activity. The next generation tools I develop will enhance our ability to dissect biological processes such as the complex neuronal network of the brain and accelerate the potential clinical use of optogenetics. My diverse team of mentors, advisors and collaborators have been chosen to both ensure my success and to further my training in the relevant areas associated with this project such as ion channel biology, chemical biology, electrophysiology, optogenetics and neuroscience. My training plan will equip me with technical skills and knowledge for developing novel chemo-optogenetic actuators for in vivo neuroscience applications and beyond, and provide a foundation for a successful transition into an independent researcher.